MS treatment de-escalation: review and commentary.

Almost all currently licensed disease-modifying therapies (DMTs) for MS treatment require prolonged if not lifelong administration. Yet, as people age, the immune system has increasingly reduced responsiveness, known as immunosenescence. Many MS DMTs reduce the responsiveness of the immune system, increasing the risks for infections and possibly cancers. As people with MS (pwMS) age, it is recognized that inflammatory MS activity declines. Several studies have addressed de-escalation of DMTs for relapsing MS under special circumstances. Here, we review evidence for de-escalating DMTs as a strategy that is particularly relevant to pwMS of older age. Treatment de-escalation can involve various strategies, such as extended or reduced dosing, switching from high-efficacy DMTs having higher risks to moderately effective DMTs with lesser risks, or treatment discontinuation. Studies have suggested that for natalizumab extended dosing maintained clinical efficacy while reducing the risk of PML. Extended interval dosing of ocrelizumab mitigated the decline of Ig levels. Retrospective and observational discontinuation studies demonstrate that age is an essential modifier of drug efficacy. Discontinuation of MS treatment in older patients has been associated with a stable disease course, while younger patients who discontinued treatment were more likely to experience new clinical activity. A recently completed 2-year randomized-controlled discontinuation study in 260 stable pwMS > 55 years found stable clinical multiple sclerosis with only a small increased risk of new MRI activity upon discontinuation. DMT de-escalation or discontinuation in MS patients older than 55 years may be non-inferior to continued treatment with immunosuppressive agents having higher health risks. However, despite several small studies, a definite conclusion about treatment de-escalation in older pwMS will require larger and longer studies. Ideally, comparison of de-escalation versus continuation versus discontinuation of DMTs should be done by prospective randomized-controlled trials enrolling sufficient numbers of subjects to allow comparisons for MS patients of both sexes within age groups, such as 55-59, 60-65, 66-69, etc. Optimally, such studies should be 3 years or longer and should incorporate testing for specific markers of immunosenescence (such as T-cell receptor excision circles) to account for differential aging of individuals.

Intrathecal administration of mesenchymal stem cells in patients with adrenomyeloneuropathy.

Background and objectives: X-linked adrenomyeloneuropathy (AMN) is an inherited neurodegenerative disorder associated with mutations in the ABCD1 gene and the accumulation of very long-chain fatty acids (VLFCAs) in plasma and tissues. Currently, there is no effective treatment for AMN. We have aimed to evaluate the therapeutic effects of mesenchymal stem cell (MSC) transplantation in patients with AMN. Methods: This is a small cohort open-label study with patients with AMN diagnosed and treated at the University Hospital in Olsztyn, Poland. All patients met clinical, biochemical, MRI, and neuropsychological criteria for AMN. MSCs derived from Wharton jelly, 20 × 106 cells, were administered intrathecally three times every 2 months, and patients were followed up for an additional 3 months. The primary outcome measures included a blinded assessment of lower limb muscle strength with the Medical Research Council Manual Muscle Testing scale at baseline and on every month visits until the end of the study. Additional outcomes included measurements of the timed 25-feet walk (T25FW) and VLFCA serum ratio. Results: Three male patients with AMN with an age range of 26-37 years participated in this study. All patients experienced increased muscle strength in the lower limbs at the end of the study versus baseline. The power grade increased by 25-43% at the baseline. In addition, all patients showed an improvement trend in walking speed measured with the T25FW test. Treatment with MSCs in patients with AMN appeared to be safe and well tolerated. Discussion: The results of this study demonstrated that intrathecal administration of WJ-MSC improves motor symptoms in patients with AMN. The current findings lend support to the safety and feasibility of MSC therapy as a potentially viable treatment option for patients with AMN.

Linking Mechanisms of Vitamin D Signaling with Multiple Sclerosis.

Environmental triggers often work via signal transduction cascades that modulate the epigenome and transcriptome of cell types involved in the disease process. Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system being characterized by a combination of recurring inflammation, demyelination and progressive loss of axons. The mechanisms of MS onset are not fully understood and genetic variants may explain only some 20% of the disease susceptibility. From the environmental factors being involved in disease development low vitamin D levels have been shown to significantly contribute to MS susceptibility. The pro-hormone vitamin D3 acts via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) as a high affinity ligand to the transcription factor VDR (vitamin D receptor) and is a potent modulator of the epigenome at thousands of genomic regions and the transcriptome of hundreds of genes. A major target tissue of the effects of 1,25(OH)2D3 and VDR are cells of innate and adaptive immunity, such as monocytes, dendritic cells as well as B and T cells. Vitamin D induces immunological tolerance in T cells and reduces inflammatory reactions of various types of immune cells, all of which are implicated in MS pathogenesis. The immunomodulatory effects of 1,25(OH)2D3 contribute to the prevention of MS. However, the strength of the responses to vitamin D3 supplementation is highly variegated between individuals. This review will relate mechanisms of individual's vitamin D responsiveness to MS susceptibility and discuss the prospect of vitamin D3 supplementation as a way to extinguish the autoimmunity in MS.

Reconsidering the route of drug delivery in refractory multiple sclerosis: Toward a more effective drug accumulation in the central nervous system.

Multiple sclerosis is a chronic demyelinating disease with different disease phenotypes. The current FDA-approved disease-modifying therapeutics (DMTs) cannot cure the disease, but only alleviate the disease progression. While the majority of patients respond well to treatment, some of them are suffering from rapid progression. Current drug delivery strategies include the oral, intravenous, subdermal, and intramuscular routes, so these drugs are delivered systemically, which is appropriate when the therapeutic targets are peripheral. However, the potential benefits may be diminished when these targets sequester behind the barriers of the central nervous system. Moreover, systemic drug administration is plagued with adverse effects, sometimes severe. In this context, it is prudent to consider other drug delivery strategies improving their accumulation in the brain, thus providing better prospects for patients with rapidly progressing disease course. These targeted drug delivery strategies may also reduce the severity of systemic adverse effects. Here, we discuss the possibilities and indications for reconsideration of drug delivery routes (especially for those "non-responding" patients) and the search for alternative drug delivery strategies. More targeted drug delivery strategies sometimes require quite invasive procedures, but the potential therapeutic benefits and reduction of adverse effects could outweigh the risks. We characterized the major FDA-approved DMTs focusing on their therapeutic mechanism and the potential benefits of improving the accumulation of these drugs in the brain.

Impact of Diminished Expression of circRNA on Multiple Sclerosis Pathomechanisms.

Circular RNA (circRNA) molecules represent a novel and unique class of endogenous non-coding RNAs controlling the expression and function of microRNA (miRNA) and post-transcriptional regulation. Recent studies implicated circRNA in the pathomechanism of multiple sclerosis (MS). Hybridization microarray was used to define the circRNA profile in the peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with relapsing-remitting MS (RRMS: 10 in relapse, 10 in remission) and 10 healthy controls (HCs). We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with an independent cohort of 45 RRMS patients (18 in relapse, 27 in remission) and 27 HCs. Microarray analysis revealed 246 circRNAs differentially downregulated (P < 0.05) in RRMS patients versus HCs. We validated two circRNAs of the three showing the lowest levels of differential expression in the RRMS remission group versus the HC group: hsa_circRNA_101145 and hsa_circRNA_001896. Their expression was significantly decreased during remission in RRMS (P = 0.0000332, FC = 0.385 and P = 0.0455, FC = 0.591, respectively) and in patients showing a lower level of disability (hsa_circRNA_101145, P = 0.0695; hsa_circRNA_001896, P = 0.0008). Bioinformatic analysis revealed 10 miRNAs interacting with these circRNAs in a complementary manner and led to the discovery of three protein-coding mRNAs downregulated in patients with RRMS during remission. These transcripts have been previously implicated in oxidative stress, blood-brain barrier permeability, microglia function, and extracellular matrix molecules altering the microenvironment and inhibiting oligodendrocyte progenitor cells. circRNAs displayed a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNAs with low expression in important mechanistic pathways of RRMS.

Fluid phase biomarkers in multiple sclerosis.

PURPOSE OF REVIEW: Multiple sclerosis (MS) is highly heterogenic disorder with respect to clinical course, diagnosis, and treatment response. There is an urgent need to search for simply and reliable fluid body biomarker which would assist the diagnosis and prediction of clinical and treatment prognosis. RECENT FINDINGS: 'Traditional' MS biomarkers, with exception of cerebrospinal fluid oligoclonal bands, still are having limited clinical value. Therefore, there is growing interest in novel molecules and ingredients. The most robust results have been generated with regard to cerebrospinal fluid and serum levels of neurofilament light chains (NfL). However, there are still some limitations related to specificity of NfL which delays its use in everyday practice. We present a new approach to search for biomarkers involving extracellular RNA, particularly microRNA (miRNA), and small extracellular vesicles. MiRNA represents an important molecular mechanism influencing gene expression, including those involved in MS pathogenesis and extracellular vesicles transfer multiple cargo, including myelin molecules from parental cells of central nervous system to the long-distance targets. SUMMARY: MiRNAs which control gene expression in cells involved in autoimmune processes in MS as well as extracellular vesicles transferring myelin content might generate a new promising categories of biomarkers of MS.

Multiple Sclerosis: circRNA Profile Defined Reveals Links to B-Cell Function.

BACKGROUND AND OBJECTIVES: To investigate the total circular RNA (circRNA) profile in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls (HCs). METHODS: Hybridization microarray was used to define the circRNA profile in peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with RRMS (10 in relapse and 10 in remission) and 10 HCs. We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-PCR with an independent cohort of 47 patients with RRMS (19 in relapse and 28 in remission) and 27 HCs. RESULTS: Microarray analysis revealed 914 transcripts to be differentially expressed between patients with RRMS in relapse and HCs (p < 0.05). We validated 3 circRNAs from 5 showing highest levels of differential expression in the RRMS relapse vs HC group: hsa_circRNA_101348, hsa_circRNA_102611, and hsa_circRNA_104361. Their expression was significantly increased during relapse in RRMS (p = 0.0002, FC = 2.9; p = 0.01, FC = 1.6; and p = 0.001, FC = 1.5, respectively) and in patients showing gadolinium enhancement on brain MRI (hsa_circRNA_101348, p = 0.0039, FC = 2.4; hsa_circRNA_104361, p = 0.029, FC = 1.7). Bioinformatic analysis revealed 15 microRNAs interacting with these circRNAs in a complementary manner and led to the discovery and validation of 3 protein-coding RNAs upregulated in patients with RRMS during relapse. Two of these, AK2 and IKZF3, have previously been implicated in B-cell function. DISCUSSION: circRNAs display a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNA in the known disturbed B-cell activity in RRMS and thus represent a novel biomarker for monitoring relapse activity.

B cell targeting therapies in MS patients during the SARS-CoV-2 pandemic - when immunosuppression meets infection?

INTRODUCTION: Research into the mechanisms of autoimmune demyelination have highlighted B cells in this process. Therapies targeting this population were a recent addition to the multiple sclerosis (MS) drugs portfolio. The SARS-CoV-2 pandemic and the risk of severe COVID-19 have challenged the safety of B cell depletion in MS patients. STATE OF THE ART: Selective depletion of B cells by monoclonal antibodies as monotherapy in MS has been shown to profoundly suppress disease activity among relapsing-remitting MS patients. Furthermore ocrelizumab, a humanised anti-CD20 monoclonal antibody, was the first licensed therapy in primary progressive MS. Based on the concept of the role of B cells in MS, many therapeutic approaches are emerging as novel ways to treat autoimmune demyelination. However, during the SARS-CoV-2 pandemic, a conservative approach toward limiting immune suppression in MS patients has been proposed. CLINICAL IMPLICATIONS: Emerging evidence does not support the notion of increased susceptibility among MS patients to the SARS-CoV-2 infection, or any predisposition toward greater severity of COVID-19. This also does not appear to be the case for MS patients undergoing B cell depletion therapies. Thus, any decision to withhold immune suppression in MS patients during the SARS-CoV-2 pandemic is probably incorrect. MS therapeutic decision-making should focus on the danger of poorly controlled autoimmune demyelination rather than perceived risks from COVID-19. FUTURE DIRECTIONS: The current pandemic highlights the need to develop more selective and safer methods of immunomodulation in MS. B cells represent several functionally different populations. Further research into the different role of these cells during autoimmune demyelination should yield better, safer strategies to control the encephalitogenic process.

The Heat Shock Protein HSP70 Promotes Th17 Genes' Expression via Specific Regulation of microRNA.

T helper cells type 17 (Th17) are orchestrators of autoimmune conditions, including multiple sclerosis (MS), but mechanisms of Th17 pathogenicity remain unknown. MicroRNAs (miRNA) are known to control T cells. To understand the function of miRNA in Th17, we have established a T cell line, EL4-TCR+, that resembles the expression pattern of the Th17 cells. Subsequently, we have evaluated the crosstalk between miRNA and Th17 genes' expression using a combination of gene expression profiling, gene expression manipulation, RNA and protein immunoprecipitation, as well as confocal microscopy. We have found that Th17-related miRNA were strongly expressed in EL4-TCR+ cells following the binding of the cluster of differentiation 3 (CD3) component of the T cell receptor (TCR). Furthermore, a specific inhibition of these miRNA resulted in downregulation of the critical Th17 genes' expression. Surprisingly, this mechanism relied on the function of the stress signal regulator heat shock protein 70 (HSP70). Upon activation, HSP70 co-localized intracellularly with miRNA processing proteins. Precipitation of HSP70 resulted in enrichment of the Th17-associated miRNA. Finally, HSP70 inhibition led to downregulation of the Th17 genes' expression and ameliorated development of autoimmune demyelination. Our study demonstrated that HSP70 facilitates specific miRNA function leading to Th17 genes' expression, a mechanism linking stress and autoimmunity.

microRNA and exosome profiling in multiple sclerosis.

New DNA sequencing technologies have uncovered non-coding RNA (ncRNA) as a major player in regulating cellular processes and can no longer be dismissed as "junk" or "dark" RNA. Among the ncRNA, microRNA (miRNA) is arguably the most extensively characterized category and a number of studies have implicated them in regulating critical functions that can influence autoimmune demyelination. Of specific interest to multiple sclerosis (MS), miRNA have been implicated in both regulating immune responses and myelination, thus making them an attractive candidate for both pharmacological intervention and as disease biomarkers. In addition, exosomes, small vesicles secreted by most cell types and present in all body fluids, have been also shown to play roles in immune signaling, inflammation and angiogenesis. Therefore, exosomes are also being explored as tools for therapeutic delivery and as biomarkers. This article reviews the recent advances in miRNA and exosome profiling in MS and experimental models.

Circular RNAs as a novel layer of regulatory mechanism in multiple sclerosis.

Multiple sclerosis (MS) is believed to be an autoimmune disease of the central nervous system (CNS) in which autoreactive immune cells recognizing myelin antigens lead to demyelination and axonal injury. Mechanisms inducing and controlling the pathogenesis of MS have not been fully elucidated. Recent studies suggest an important role of epigenetic processes during the development of MS. One of the most significant discoveries in the field of epigenetic contribution to immune response has been the recognition of a group of microRNAs (miRNAs). These single-stranded non-coding RNA molecules regulate the expression of genes encoding proteins and have already been shown to be involved in pathogenesis of MS. Some miRNAs enhance generation of pro-inflammatory immune cells by promoting Th1 and Th17 pathways and others contribute to regulatory and tissue repair processes. The miRNA-dependent controlling process of autoimmune reactions is highly complex because of miRNA redundancy and multitarget nature of most of these molecules. Recently it was discovered that circular RNAs (circRNA) representing a new class of RNA possess a unique ability to control miRNAs by blocking their activity. CircRNAs are called natural miRNA "sponges" as the single circRNA molecule is able to neutralize several miRNAs and thus might determine the availability of miRNAs for their posttranscription regulation. Thus, circRNAs emerged as critical factors in epigenetic regulation of many human diseases including MS. In addition, in contrary to other RNA species they are very stable in the blood and other biological fluids and thus might be considered as a candidate for a biomarker of MS.

Multiple sclerosis: Serum-derived exosomes express myelin proteins.

BACKGROUND: Exosomes are small extracellular vesicles that provide cell-to-cell communication and are involved in immunoregulation. OBJECTIVE: To investigate serum exosomes for the presence of myelin proteins outside the central nervous system (CNS) and their role in multiple sclerosis (MS). METHODS: Serum, cerebrospinal fluid (CSF), and peripheral blood mononuclear cell (PBMC) samples were collected from 45 patients with relapsing-remitting MS (RRMS), 30 patients with secondary progressive MS (SPMS), and 45 healthy controls. Exosomes were isolated using a polymer formulation method, and their size, concentration, and CNS myelin protein contents were measured by a nanoparticle tracking analysis, enzyme-linked immunosorbent assays, and Western blot. RESULTS: We found that exosomes expressed three major myelin proteins, myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG). Exosomal content of MOG strongly correlated with disease activity and was highest in RRMS patients in relapse and in SPMS patients. Serum-derived exosomes induced proliferation of MOG-T cell receptor transgenic T cells confirming that serum exosomes maintained MOG immunogenicity. CONCLUSION: Exosomes isolated outside CNS tissue expressed myelin proteins, and the presence of MOG correlated strongly with disease activity. We conclude that exosomes might enhance and/or perpetuate anti-myelin immune reactions in MS and may provide novel markers of disease activity.

Global exosome transcriptome profiling reveals biomarkers for multiple sclerosis.

OBJECTIVE: Accumulating evidence supports a role for exosomes in immune regulation. In this study, we investigated the total circulating exosome transcriptome in relapsing-remitting multiple sclerosis (RRMS) patients and healthy controls (HC). METHODS: Next generation sequencing (NGS) was used to define the global RNA profile of serum exosomes in 19 RRMS patients (9 in relapse, 10 in remission) and 10 HC. We analyzed 5 million reads and >50,000 transcripts per sample, including a detailed analysis of microRNAs (miRNAs) differentially expressed in RRMS. The discovery set data were validated by quantification using digital quantitative polymerase chain reaction with an independent cohort of 63 RRMS patients (33 in relapse, 30 in remission) and 32 HC. RESULTS: Exosomal RNA NGS revealed that of 15 different classes of transcripts detected, 4 circulating exosomal sequences within the miRNA category were differentially expressed in RRMS patients versus HC: hsa-miR-122-5p, hsa-miR-196b-5p, hsa-miR-301a-3p, and hsa-miR-532-5p. Serum exosomal expression of these miRNAs was significantly decreased during relapse in RRMS. These miRNAs were also decreased in patients with a gadolinium enhancement on brain magnetic resonance imaging. In vitro secretion of these miRNAs by peripheral blood mononuclear cells was also significantly impaired in RRMS. INTERPRETATION: These data show that circulating exosomes have a distinct RNA profile in RRMS. Because putative targets for these miRNAs include the signal transducer and activator of transcription 3 and the cell cycle regulator aryl hydrocarbon receptor, the data suggest a disturbed cell-to-cell communication in this disease. Thus, exosomal miRNAs might represent a useful biomarker to distinguish multiple sclerosis relapse. Ann Neurol 2017;81:703-717.

The role of exosomes in CNS inflammation and their involvement in multiple sclerosis.

Multiple sclerosis (MS) is a putative autoimmune disease of the central nervous system (CNS) in which autoreactive immune cells recognizing myelin antigens lead to demyelination and axonal injury. Mechanisms relevant to the pathogenesis of MS have not been fully elucidated, particularly those underlying initiation of immune system dysfunction. For example, it is not known how reactivity against CNS components is generated within the peripheral immune system. In this review, we propose that a significant contribution to the immunoregulatory events may derive from a cell-to-cell communication system involving the production, secretion and transfer of extracellular vesicles known as exosomes. Herein, we discuss in detail the biogenesis and roles of these cell surface-generated vesicles from the standpoint of receptors and their cargo, microRNA. It is well known that exosomes can cross the blood-brain barrier and thus may contribute to the spread of brain antigens to the periphery. Further understanding of exosome-dependent mechanisms in MS should provide a novel angle to the analysis of the pathogenesis of this disease. Finally, we launch the idea that exosomes and their contents may serve as biomarkers in MS.

miR-155-3p Drives the Development of Autoimmune Demyelination by Regulation of Heat Shock Protein 40.

microRNA-155 (miR-155) plays an important role in posttranscriptional gene regulation of the immune system. We and others have described miR-155 upregulation in T helper cells (Th) during the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We have shown that mice in which the miR-155 host gene (MIR155HG) has been deactivated are resistant to EAE. MIR155HG produces two different miRNA strands, miR-155-5p and miR-155-3p, and miR-155-5p has been considered the only functional miR-155 form. Surprisingly, we found that miR-155-3p is also strongly upregulated in Th cells infiltrating the brain in EAE. Functional manipulation of miR-155-3p expression revealed its particular role in regulation of Th17 development. The search for miRNA-155-3p target genes highlighted transcripts of two heat shock protein 40 genes, Dnaja2 and Dnajb1. These two genes negatively regulated Th17 differentiation, leading to decreased EAE. Therefore, our findings provide new insights into a previously unknown mechanism by which miR-155-3p controls Th17 cell differentiation and autoimmune demyelination. SIGNIFICANCE STATEMENT: Multiple sclerosis (MS) is brain-specific autoimmune disease mediated by T helper (Th) cells autoreactive to myelin. The mechanisms leading to MS are not fully understood and microRNAs (miRNAs) emerge as important regulators of the process. We report that, in an MS murine model of experimental autoimmune encephalomyelitis, miR-155 controls Th cell function by an unusual mechanism involving a rare form, miR-155-3p. miR-155-3p is specifically found in brain-infiltrating myelin-autoreactive CD4(+) T cells and contributes to the development of an encephalitogenic Th17 population. miR-155-3p promotes Th17 by inhibiting two heat shock protein 40 genes, Dnaja2 and Dnajb1. Our findings indicate a unique miRNA function in the brain-infiltrating Th cells and suggest Dnaja2 and Dnajb1 as targets for intervention in autoimmune demyelination.

Heat shock protein 70 (Hsp70) interacts with the Notch1 intracellular domain and contributes to the activity of Notch signaling in myelin-reactive CD4 T cells.

Notch receptors (Notch1-4) are involved in the differentiation of CD4 T cells and the development of autoimmunity. Mechanisms regulating Notch signaling in CD4 T cells are not fully elucidated. In this study we investigated potential crosstalk between Notch pathway molecules and heat shock protein 70 (Hsp70), the major intracellular chaperone involved in the protein transport during immune responses and other stress conditions. Using Hsp70(-/-) mice we found that Hsp70 is critical for up-regulation of NICD1 and induction of Notch target genes in Jagged1- and Delta-like1-stimulated CD4 T cells. Co-immunoprecipitation analysis of wild-type CD4 T cells stimulated with either Jagged1 or Delta-like1 showed a direct interaction between NICD1 and Hsp70. Both molecules co-localized within the nucleus of CD4 T cells stimulated with Notch ligands. Molecular interaction and nuclear colocalization of NICD1 and Hsp70 were also detected in CD4 T cells reactive against myelin oligodendrocyte glycoprotein (MOG)35-55, which showed Hsp70-dependent up-regulation of both NICD1 and Notch target genes. In conclusion, we demonstrate for the first time that Hsp70 interacts with NICD1 and contributes to the activity of Notch signaling in CD4 T cells. Interaction between Hsp70 and NICD1 may represent a novel mechanism regulating Notch signaling in activated CD4 T cells.

Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination.

MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS.

Brain glycolipids suppress T helper cells and inhibit autoimmune demyelination.

The CNS is considered an immune privileged site because its repertoire of highly immunogenic molecules remains unseen by the immune system under normal conditions. However, the mechanism underlying the inhibition of immune reactions within the CNS environment is not known, particularly in regions containing myelin, which contains several potent proteins and lipids that are invariably recognized as foreign by immune system cells. Sulfatides constitute a major component of myelin glycolipids and are known to be capable of raising an immune response. In this study, the effect of sulfatides on mouse T cell function and differentiation was analyzed in vitro and in vivo. We found profound inhibition of sulfatide-dependent T cell proliferation which was particularly pronounced in naive T helper (Th) cells. The inhibitory effect of sulfatides on T cell function was CD1d-independent and was not related to apoptosis or necrosis but did involve the induction of anergy as confirmed by the upregulation of early growth response 2 transcription factor. A glycolipid 3-sulfate group was essential for the T cell suppression, and the T cell inhibition was galectin-4-dependent. Sulfatide stimulation in vitro led to prominent suppression of Th17 differentiation, and this was related to a decrease in susceptibility to disease in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. Thus, we have defined a novel mechanism of negative regulation of T cell function by endogenous brain-derived glycolipids, a family of molecules traditionally deemphasized in favor of myelin proteins in studies of CNS autoimmunity.

Plasmocytoid dendritic cell deficit of early response to toll-like receptor 7 agonist stimulation in multiple sclerosis patients.

Plasmacytoid dendritic cells (pDCs), an important immunoregulatory population, are characterized by vigorous secretion of type I interferons (IFNs) in response to toll-like receptor (TLR) 7 and 9 stimulation. We studied the function of pDCs in multiple sclerosis (MS) patients by analysis of TLR7 responses. We assessed a pDC secretion pattern of cytokines in the short term PBMC cultures stimulated with TLR7 agonist. pDCs sorted from PBMCs of both MS patients and controls were used to assess TLR7 expression profile. TLR7 induced signaling in pDCs has been analyzed with intracellular flow cytometry. We have identified a clinically correlated significant decrease of the TLR7-induced IFN-alfa (IFNa) secretion by pDCs from MS patients. This deficit has been accompanied by insufficient intracellular phosphorylation of protein kinase Akt and a decrease of the TLR7 gene expression in MS pDCs. Our results demonstrated a selective pDC deficit in MS supporting a relationship between pDCs and mechanisms of MS.